The SR Protein SC35 Is Responsible for Aberrant Splicing of the E1α Pyruvate Dehydrogenase mRNA in a Case of Mental Retardation with Lactic Acidosis

Abstract

Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leigh's encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1␣ PDH gene. Recently, we identified a novel mutation of the E1␣ PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1␣ PDH mRNA. Our findings open the exciting prospect for a novel therapy of an inherited disease by altering the level of a specific splicing factor.Removal of intervening sequences (introns) from precursor messenger RNAs (pre-mRNA) is an essential step in the expression of most metazoan protein-encoding genes, often regulated in a cell-type-specific or developmental manner. Alternative splicing enables the same precursor to give rise to several mRNAs that code for proteins having distinct functions. Thus, the precise selection of 5Ј and 3Ј splicing sites is a way to generate diversity and may lead to the regulation of gene expression according to tissue type or during development of the organism. The results of a recently published genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays (22) indicate that at least 74% of human multiexon genes are alternatively spliced.Among the splicing factors involved in splice site choice, members of the SR protein family have been extensively studied (see references 18 and 33 for reviews). SR proteins are characterized by the presence of one or two copies of an RNA recognition motif and a carboxyl-terminal domain rich in arginine and serine residues (RS domain). The RS domain is responsible for specific protein-protein interactions between RS domain-containing proteins (25,(42)(43)(44), whereas the RNA recognition motif domain recognizes several classes of specific RNA motifs, including exonic splicing enhancers (ESEs) and intronic splicing enhancers. These sequences have been demonstrated to play a key role in both alternative and constitutive splice site selection (see references 2 and 41 for reviews). This activity is counteracted by that of splicing repressors, such as members of the hnRNP family which can bind RNA in a nonspecific way but also recognize negative regulatory elements known as exonic and intronic splicing silencers (see references 29 and 43 for reviews). Such an antagonism accounts for the ability of SR proteins to influence splice site choice in a concentration-dependent manner (28, 33).

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

“…The G to A change at position 26 generates an ESE that serves as a binding site for SC35 and thereby activates a cryptic 5Ј splice site 20 nucleotides downstream of the mutation within the same intron (ref. 25 and Fig. 4A).…”

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

“…M-019711-00-0005) consisting of four siRNAs or a single siRNA whose efficacy has been previously demonstrated (37). SMARTpool siRNAs were also used to deplete SRSF3 (product no.…”

Ambulatory Blood Pressure Is Associated With Polymorphic Variation in P2X Receptor Genes

“…Genetic variants altering SC35 binding sites can have large effects on gene expression. 32 Additional studies to quantify the effect of the associated SNPs and of additional SNPs (particularly in the 24-kb regions between the 2 genes, which was not covered with tagSNPs in this study) on the expression of both P2X7 and P2X4 may allow the detection of regulatory effects mediating BP differences.…”